Reagents and methods for identifying, enriching, and/or expanding antigen-specific t cells

ABSTRACT

Antigen-specific T cells, including nave T cells, and including rare precursor cells are enriched and expanded in culture. Enrichment and expansion provides a platform for more effective immunotherapy by adoptive transfer, as well as platforms for personalizing immunotherapy by determining T cell reactivity with a library of candidate peptide antigens.

TECHNICAL FIELD

The subject matter of this application relates to immunotherapy, and isrelated to the subject matter disclosed in PCT/US2014/25889 filed Mar.13, 2014, the contents of which are incorporated by reference.

BACKGROUND

Expansion of antigen-specific T cells is complicated by the rarity ofantigen-specific naive precursors, which can be as few as one permillion. To generate the large numbers of tumor-specific T cells (forexample) required for adoptive therapy, lymphocytes are conventionallystimulated with antigen over many weeks, often followed by T cellselection and sub-cloning in a labor intensive process.

There is a need for technologies that can quickly generate large numbersand high frequencies of antigen-specific T cells from naive precursors,or quickly identify T cell responses to candidate peptide antigens, forboth therapeutic and diagnostic purposes.

SUMMARY OF THE INVENTION

In various aspects, the invention provides for rapid enrichment andexpansion of antigen-specific T cells in culture, including from naïve Tcells, and including from rare precursor cells. The invention therebyprovides a platform for more effective immunotherapy (e.g., by adoptivetransfer). The invention further provides platforms for rapidlyidentifying antigen-specific T cell responses from patient lymphocytes,and platforms for personalizing immunotherapy, by determining T cellreactivity against a library of candidate peptide antigens.

The invention employs in various embodiments nano-scale artificialAntigen Presenting Cells (aAPCs), which capture and deliver stimulatorysignals to immune cells, such as antigen-specific T lymphocytes, such asCTLs. Signals present on the aAPCs can include Signal 1, antigenicpeptide presented in the context of Major Histocompatibility Complex(MHC) (class I or class II); and Signal 2, one or more co-stimulatoryligands that modulate T cell response. Signal 2 in some embodiments is aligand that binds and activates through CD28. In various embodiments,the particle material is paramagnetic and is preferably biocompatible,such as dextran-coated iron oxide particles. Paramagnetic particlesallow for magnetic capture or “enrichment” by application of a magneticfield, as well as activation and subsequent expansion ofantigen-specific lymphocytes within the enriched cell fraction, which isalso enhanced by application of a magnetic field.

In some aspects, the invention provides a method for preparing anantigen-specific T-cell population. The method comprises providing asample comprising T-cells from a patient. In some embodiments thepatient is in need of adoptive transfer of antigen-specific T-cells. Thesample may be a PBMC sample, or sample obtained by leukapheresis. Thesample can be enriched for T cells of interest, such as CD8+ T cellsand/or naïve T cells. The sample containing the T cells is contactedwith a population of paramagnetic aAPCs presenting antigens that arecommon for the disease of interest (e.g., tumor-type), and/or presentingone or more antigens selected on a personalized basis. In certainembodiments, each aAPC bead presents a single antigen, and a cocktail ofaAPC beads each presenting different antigens is used forenrichment/expansion. In some embodiments, from about 3 to about 10different antigens are presented by the antigen presenting complexes.The paramagnetic property is used to capture or “enrich” the sample forantigen-specific T-cells, by placing a magnet within proximity tothereby separate aAPC-associated cells from non-associated cells.Recovered T-cells can be expanded in vitro by culture with the aAPCs,and expansion of antigen-specific cells is further enhanced by thepresence of a magnetic field. The enrichment and expansion process maybe repeated one or more times, for optimal expansion (and furtherpurity) of antigen-specific cells.

In certain embodiments, the method provides for about 1000-10,000 foldexpansion (or more) of antigen-specific T cells, with more than about10⁸ antigen-specific T cells being generated in the span of, forexample, 1-3 weeks. The resulting cells can be administered to thepatient to treat disease. Antigen-specific frequency is an independentlyimportant parameter for optimal expansion after transfer, sincecompetition for growth signals from irrelevant, co-transferred cells maysignificantly attenuate homeostatic expansion of anti-tumor T cells ofinterest. In some embodiments, the methods further compriseadministering at least about 106 antigen-specific T cells to a patientrecipient as adoptive immunotherapy.

In still other aspects, the invention provides methods for selecting Tcell antigens on a personalized basis. For example, an array or libraryof aAPCs each presenting a candidate antigenic peptide, is screened withT cells from a subject or patient, and the response of the T cells toeach aAPC-peptide is determined or quantified. T cell responses can bequantified, for example, by cytokine expression or expression of othersurrogate marker of T cell activation. Exemplary assays platformsinclude immunochemistry, such as ELISA, or amplification of expressedgenes, e.g., by RT-PCR. In other embodiments, T cell activation isquantified by measuring an intracellular signaling event that isindicative of T cell activation, such as calcium signaling. Such assayscan employ any variety of colorimetric assays known in the art.

Peptide antigens showing the most robust responses are selected forimmunotherapy, including in some embodiments adoptive immunotherapy,which may be achieved through enrichment and expansion ofantigen-specific T cells. In some embodiments, and particularly forcancer immunotherapy, a patient's tumor is genetically analyzed, andtumor antigens are predicted from the patient's unique tumor mutationsignature. These predicted antigens (“neoantigens”) are synthesized andscreened against the patient's T cells using the aAPC platform. Oncereactive antigens are identified/confirmed, aAPCs can be prepared forthe enrichment and expansion protocol described herein, or the aAPCs canbe directly administered to the patient in some embodiments.

In some embodiments, a patient or subject's T cells are screened againstan array or library of paramagnetic aAPCs, each presenting a differentcandidate peptide antigen. This screen can provide a wealth ofinformation concerning the subject or patient's T cell repertoire, andthe results are useful for diagnostic or prognostic purposes. Forexample, the number and identity of T cell anti-tumor responses againstmutated proteins, overexpressed proteins, and/or other tumor-associatedantigens can be used as a biomarker to stratify risk, to monitorefficacy of immunotherapy, or predict outcome of immunotherapytreatment. Further, the number or intensity of such T cell responses maybe inversely proportionate to the risk of disease progression or may bepredictive of resistance or non-responsiveness to chemotherapy. In otherembodiments, a subject's or patient's T cells are screened against anarray or library of nano-APCs each presenting a candidate peptideantigen, and the presence of T cells responses, or the number orintensity of these T cells responses, provides information concerningthe health of the patient, for example, by identifying autoimmunedisease, or identifying that the patient has a sub-clinical tumor. Inthese embodiments, the process not only identifies a potential diseasestate, but provides an initial understanding of the disease biology.

The present invention thereby provides for diagnostic and therapeuticadvances in a number of T cell-related diseases or conditions, includingcancer, autoimmune disease, and other diseases in which detection,enrichment, activation, and/or expansion of antigen-specific immunecells ex vivo is therapeutically or diagnostically desirable.

Further aspects and embodiments of the invention will be apparent to theskilled artisan based on the following detailed description.

DESCRIPTION OF THE FIGURES

FIGS. 1A-B present schematics of an embodiment for enrichment andexpansion of antigen-specific T cells. (FIG. 1A) an embodiment of ananoscale artificial antigen presenting cell (nano-aAPC) is synthesizedby coupling MHC-Ig dimer (Signal 1) and a co-stimulatory anti-CD28antibody (Signal 2) to a 50-100 nm iron-dextran nanoparticle. (FIG. 1B)Schematic of magnetic enrichment. Antigen-specific CD8+ T cells bound tonano-aAPC are retained in a magnetic column in the “enrichment” step,while non-cognate cells are less likely to bind.

Enriched T cells are then activated by nano-aAPC and proliferate in the“expansion” step.

FIGS. 2A-2C. Nano-aAPC-Mediated Enrichment of Antigen-Specific T Cells.(FIG. 2A) Nano-aAPC mediate antigen-specific enrichment of cognate,Thy1.1+ pmel cells from a pool of thousand-fold more polyclonal, Thy1.2+B6 splenocytes. (FIG. 2B) Summary of antigen-specific cell frequency andpercent cells recovered after pmel enrichment performed as in (FIG. 2A)with increasing amounts of nano-aAPC. (FIG. 2C) Enrichment of endogenousDb-gp100 splenocytes by nano-aAPC (top). Frequency of non-cognateKb-Trp2 cells does not increase after enrichment (bottom).

FIGS. 3A-3G. Expansion of Antigen-Specific T cells After Enrichment.(FIG. 3A) Schematic of cell fractions used to assess the effect ofenrichment on expansion. Particle-bound antigen-specific T cells arecaptured in a magnetic column (positive fraction), whereas unbound cellspass through (negative fraction). The negative fraction can be addedback to the positive fraction to undo the effect of enrichment(positive+negative). (FIG. 3B) Increased frequency of antigen-specificcells generated after seven days of culture as a result of enrichmentwith Kb-Trp2 nano-aAPC. Negative (left), positive (middle) andpositive+negative (right) fractions were cultured for seven days, thenstained with cognate Kb-Trp2 (top) and control Kb-SIINF (bottom) dimer.(FIG. 3C) 10-15 fold increase in frequency of Kb-Trp2 cells (*, p<0.001by t-test) when cells are enriched. (FIG. 3D) Representative FACS plotsof Db-gp100, Kb-SIINF, and Ld-A5 nano-aAPC expansion seven days afterenrichment with cognate nano-aAPC. (FIG. 3E) Summary of percentantigen-specific cells (left) and total antigen specific cells (right)after enrichment and activation with indicated nano-aAPC. (FIG. 3F)Three antigens (Db-gp100, Kb-SIINF, Kb-Trp2) enriched and expandedsimultaneously. Representative FACS plots of antigen-specificity foreach antigen from the same T cell culture. (FIG. 3G) Comparison ofantigen-specificity (left) and total antigen-specific cells (right)generated for the three indicated antigens when enriched and expandedindividually or together.

FIGS. 4A-4D: Adoptive Transfer of enriched and expanded T Cells MediatesTumor Rejection. (FIG. 4A) Effect of lymphodepletion and decreasedbystander competition on expansion after adoptive transfer. B6 mice wereuntreated or lymphodepleted with 500 cGy gamma radiation one day priorto adoptive transfer of 10⁵ pmel T cells in the presence of either 10⁶or 10⁷ irrelevant B6 cells. Both lymphodepletion and administration offewer bystander cells increased the frequency of pmel T cells recoveredfrom spleen and lymph nodes (p<0.01 by two-way ANOVA). (FIG. 4B) Totalnumber of Thy1.1+ pmel cells recovered in (FIG. 4A). (FIG. 4C) Kb-Trp2and Db-gp100 Enriched+Expanded lymphocytes cultured for 7 days prior toadoptive transfer inhibited melanoma growth (p<0.01 by two-way ANOVA, 8mice/group). Mice were injected with subcutaneous melanoma eight daysprior and irradiated with 500 cGy gamma irradiation one day prior.Non-cognate enriched and expanded lymphocytes (SIINF) did not inhibittumor growth (compared to untreated), whereas cognate enriched andexpanded (Trp2+gp100) did. (FIG. 4D) Survival of animals from (FIG. 4C).2/8 mice showed complete rejection of tumors in the Kb-Trp2 and Db-gp100treated group, which had significantly longer survival compared tonon-cognate and untreated groups (p<0.01 by Mantel-Cox).

FIGS. 5A-5B: Expansion of Human Anti-Tumor Response. CD8+ PBMCs wereisolated from healthy donors and expanded using the enrichment andexpansion protocol for one week. (FIG. 5A) Representative staining andfrequency of A2-NY-ESO1 (top) and A2-MART1 (bottom) specific cellsimmediately after CD8 isolation (Day 0, left) and after one week ofenrichment and expansion (Day 7, right). (FIG. 5B) Summary of percentantigen-specific cell frequency (top) and total antigen specific cells(bottom) after enrichment/expansion with indicated nano-aAPC. Resultsderived from three experiments with different donors.

FIGS. 6A-6C: Neo-Epitope Expansion. (FIG. 6A) Schematic of process forgenerating candidate peptides for B16 and CT26 mutomes. 17-mer sequencessurrounding single-base pair substitutions (SBS) are assessed for MHCbinding by MHCNet prediction algorithm. (FIG. 6B) Representative bindingof cells expanded with nano-aAPC E+E for seven days against neo-epitopesto cognate (top) and non-cognate (bottom) MHC. FIG. (6C) Totalneo-epitope specific cells obtained at one week after E+E.

FIGS. 7A-7C: Micro-aAPC Are Not Effective For Antigen-SpecificEnrichment. (FIG. 7A) Binding of Micro- (top) and Nano- (bottom) aAPC tocognate pMEL (red) or non-cognate 2C (blue) CD8+ T cells, characterizedby fluorescent labeling of bound beads. No bead (grey) background isshown as control. (FIG. 7B) Micro-aAPC do not enrich cognate cells.Thy1.1+ pmel cells were incubated at a 1:1000 ratio with polyclonal,Thy1.2+ B6 splenocytes, and enrichment was attempted using Db-GP100microparticles. Frequency of Thy1.1+ cells did not significantlyincrease after enrichment. (FIG. 7C) Antigen-specific cell frequency andpercent of cells recovered, performed as in (FIG. 7C with increasingamounts of micro-aAPC.

DETAILED DESCRIPTION OF THE INVENTION

Adoptive immunotherapy involves the activation and expansion of immunecells ex vivo, with the resulting cells transferred to the patient totreat disease, such as cancer. Induction of antigen-specific cytotoxic(CD8+) lymphocyte (CTL) responses, for example, through adoptivetransfer could be an attractive therapy, if sufficient numbers andfrequency of activated and antigen-specific CTL can be generated in arelatively short time, including from rare precursor cells. Thisapproach in some embodiments could even generate long-term memory thatprevents recurrence of disease. In addition to cancer immunotherapy, andimmunotherapies involving CTLs, the invention finds use with otherimmune cells, including CD4+ T cells and regulatory T cells, and thus isbroadly applicable to immunotherapy for infectious disease andauto-immune disease, among others.

In one aspect, the present invention provides artificial AntigenPresenting Cells (aAPCs), which capture and deliver stimulatory signalsto immune effector cells, such as antigen-specific T lymphocytes, suchas CTLs. In some embodiments, these aAPCs offer a powerful tool foradoptive immunotherapy. Signals present on the aAPCs that support T cellactivation include Signal 1, antigenic peptide presented in the contextof Major Histocompatibility Complex (MHC), class I or class II, andwhich bind antigen-specific T-cell Receptors (TCR); and Signal 2, one ormore co-stimulatory ligands that modulate T cell response. In someembodiments of this system, Signal 1 is conferred by a monomeric,dimeric or multimeric MHC construct. A dimeric construct is created insome embodiments by fusion to a variable region or C_(H)1 region of animmunoglobulin heavy chain sequence. The MHC complex is loaded with oneor more antigenic peptides, and Signal 2 is either B7.1 (the naturalligand for the T cell receptor CD28) or an activating antibody againstCD28. Both ligands may be directly chemically coupled to the surface ofa microscale (4.5 μm) or nanoscale bead to create an artificial AntigenPresenting Cell (aAPC). In some embodiments, the particle material isparamagnetic, allowing for magnetic capture or “enrichment” byapplication of a magnetic field, as well as subsequent expansion ofantigen-specific lymphocytes within the enriched cell fraction, which isalso enhanced by application of a magnetic field. In other embodiments,the paramagnetic property supports a rapid T cell response (e.g.,activation), even from naïve cells, which can be detected within minutesto hours in vitro.

In some aspects, the invention provides a method for preparing anantigen-specific T-cell population for adoptive transfer. The methodcomprises providing a sample comprising T-cells from a patient, wherethe patient is in need of adoptive transfer of antigen-specific T-cells.The T cells, or sample containing the T cells, are contacted with apopulation of paramagnetic aAPCs as described in detail herein, each ofwhich presents a peptide antigen of interest in the context of MHC(class I or II), and thereby binds antigen-specific T-cells in thesample (including naïve antigen-specific cells that are infrequentlyrepresented). The aAPCs may present antigens that are common for thedisease of interest (e.g., tumor-type), or may present one or moreantigens selected on a personalized basis. The paramagnetic property canbe used to capture or “enrich” the sample for antigen-specific T-cells,for example, by using a magnet to separate aAPC-associated cells fromnon-associated cells. Recovered T-cells, for example, those that remainassociated with the paramagnetic aAPC particles, can be expanded invitro in the presence of the aAPCs, and expansion of antigen-specificcells is further enhanced by the presence of a magnetic field. Withoutwishing to be bound by theory, it is believed that the paramagneticaAPCs bound to the antigen-specific T cells will facilitate T cellreceptor clustering in the presence of a magnetic field. The expansionstep can proceed from about 3 days to about 2 weeks in some embodiments,or about 5 days to about 10 days (e.g., about 1 week). The enrichmentand expansion process may then be repeated one or more times, foroptimal expansion (and further purity) of antigen-specific cells. Forsubsequent rounds of enrichment and expansion, additional aAPCs may beadded to the T cells to support expansion of the larger antigen-specificT cell population in the sample. In certain embodiments, the final round(e.g., round 2, 3, 4, or 5) of expansion occurs in vivo, wherebiocompatible nanoAPCs are added to the expanded T cell population, andthen infused into the patient.

In certain embodiments, the method provides for about 1000-10,000 foldexpansion (or more) of antigen-specific T cells, with more than about10⁸ antigen-specific T cells being generated in the span of, forexample, less than about one month, or less than about three weeks, orless than about two weeks, or in about one week. The resulting cells canbe administered to the patient to treat disease. The aAPC may beadministered to the patient along with the resulting antigen-specific Tcell preparation in some embodiments.

In still other aspects, the invention provides methods for selecting Tcell antigens on a personalized basis. For example, in certainembodiments an array or library of aAPCs each presenting a candidateantigenic peptide, is screened with T cells from a subject or patient(and in the presence of a magnetic field in some embodiments), and theresponse of the T cells to each aAPC-peptide is determined orquantified. T cell response can be quantified molecularly in someembodiments, for example, by quantifying cytokine expression orexpression of other surrogate marker of T cell activation (e.g., byimmunochemistry or amplification of expressed genes such as by RT-PCR).In some embodiments, the quantifying step is performed between about 15hours and 48 hours in culture. In other embodiments, T cell response isdetermined by detecting intracellular signaling (e.g., Ca2+ signaling,or other signaling that occurs early during T cell activation), and thuscan be quantified within about 15 minutes to about 5 hours (e.g., withinabout 15 minutes to about 2 hours) of culture with the nano-aAPCs.Peptides showing the most robust responses are selected forimmunotherapy, including in some embodiments the adoptive immunotherapyapproach described herein. In some embodiments, and particularly forcancer immunotherapy, a patient's tumor is genetically analyzed (e.g.,using next generation sequencing), and tumor antigens are predicted fromthe patient's unique tumor mutation signature. These predicted antigens(“neoantigens”) are synthesized and screened against the patient's Tcells using the aAPC platform described herein. Once reactive antigensare identified/confirmed, aAPCs can be prepared for the enrichment andexpansion protocol described herein, or the aAPCs can be directlyadministered to the patient in some embodiments. In some embodiments,the antigen-presenting complex presents an antigen or a neoantigenpredicted from genetic analysis of the patient's tumor, wherein theneoantigen is formed by a mutation selected from the group consisting ofa passenger mutation, a driver mutation, an oncogene forming mutation,and a tumor suppressor destroying mutation.

In some aspects, a subject or patient's T cells are screened against anarray or library of paramagnetic nano-aAPCs (as described herein), whereeach paramagnetic nano-aAPC presents a peptide antigen. T cell responsesto each are determined or quantified as described herein, providinguseful information concerning the patient's T cell repertoire, and hencethe condition of the subject or patient.

For example, the number and identity of T cell anti-tumor responsesagainst mutated proteins, overexpressed proteins, and/or othertumor-associated antigens can be used as a biomarker to stratify risk,and in some embodiments can involve a computer-implemented classifieralgorithm to classify the response profile for drug resistance or drugsensitivity, or stratify the response profile as a candidate forimmunotherapy (e.g., checkpoint inhibitor therapy or adoptive T celltransfer therapy). For example, the number or intensity of such T cellresponses may be inversely proportionate to a high risk of diseaseprogression, and/or may directly relate to the patient's likely responseto immunotherapy, which may include one or more of checkpoint inhibitortherapy, adoptive T cell transfer, or other immunotherapy for cancer.

In still other aspects and embodiments, the patient's T cells arescreened against an array or library of paramagnetic nano-APCs, eachpresenting a candidate peptide antigen. For example, the array orlibrary may present tumor-associated antigens, or may presentauto-antigens, or may present T cell antigens relating to variousinfectious diseases. By incubating the array or library with thepatient's T cells, and in the presence of a magnetic field to encourageT cell receptor clustering, the presence of T cells responses, and/orthe number or intensity of these T cells responses, can be rapidlydetermined. This information is useful for diagnosing, for example, asub-clinical tumor, an autoimmune or immune disease, or infectiousdisease, and can provide an initial understanding of the diseasebiology, including, potential pathogenic or therapeutic T cells, T cellantigens, and an understanding of the T cell receptors of interest,which represent drug or immunotherapy targets.

Various alternative embodiments of the various aspects of the inventionare described in detail below.

The present invention provides for immunotherapy for cancer and otherdiseases in which detection, enrichment and/or expansion ofantigen-specific immune cells ex vivo is therapeutically ordiagnostically desirable. The invention is generally applicable fordetection, enrichment and/or expansion of antigen-specific T cells,including cytotoxic T lymphocytes (CTLs), helper T cells, and regulatoryT cells.

In some embodiments, the patient is a cancer patient. The enrichment andexpansion of antigen-specific CTLs ex vivo for adoptive transfer to thepatient provides for a robust anti-tumor immune response. Cancers thatcan be treated or evaluated according to the methods include cancersthat historically illicit poor immune responses or have a high rate ofrecurrence. Exemplary cancers include various types of solid tumors,including carcinomas, sarcomas, and lymphomas. In various embodimentsthe cancer is melanoma (including metastatic melanoma), colon cancer,duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductalcancer, hepatic cancer, pancreatic cancer, renal cancer, endometrialcancer, testicular cancer, stomach cancer, dysplastic oral mucosa,polyposis, head and neck cancer, invasive oral cancer, non-small celllung carcinoma, small-cell lung cancer, mesothelioma, transitional andsquamous cell urinary carcinoma, brain cancer, neuroblastoma, andglioma. In some embodiments, the cancer is a hematological malignancy,such as chronic myelogenous leukemia, childhood acute leukemia,non-Hodgkin's lymphomas, chronic lymphocytic leukemia, malignantcutaneous T-cells, mycosis fungoids, non-MF cutaneous T-cell lymphoma,lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, anddiscoid lupus erythematosus.

In various embodiments, the cancer is stage I, stage II, stage III, orstage IV. In some embodiments, the cancer is metastatic and/orrecurrent. In some embodiments, the cancer is preclinical, and isdetected in the screening system described herein (e.g., colon cancer,pancreatic cancer, or other cancer that is difficult to detect early).

In some embodiments, the patient has an infectious disease. Theinfectious disease may be one in which enrichment and expansion ofantigen-specific immune cells (such as CD8+ or CD4+ T cells) ex vivo foradoptive transfer to the patient could enhance or provide for aproductive immune response. Infectious diseases that can be treatedinclude those caused by bacteria, viruses, prions, fungi, parasites,helminths, etc. Such diseases include AIDS, hepatitis, CMV infection,and post-transplant lymphoproliferative disorder (PTLD). CMV, forexample, is the most common viral pathogen found in organ transplantpatients and is a major cause of morbidity and mortality in patientsundergoing bone marrow or peripheral blood stem cell transplants. Thisis due to the immunocompromised status of these patients, which permitsreactivation of latent virus in seropositive patients or opportunisticinfection in seronegative individuals. A useful alternative to thesetreatments is a prophylactic immunotherapeutic regimen involving thegeneration of virus-specific CTL derived from the patient or from anappropriate donor before initiation of the transplant procedure. PTLDoccurs in a significant fraction of transplant patients and results fromEpstein-Barr virus (EBV) infection. EBV infection is believed to bepresent in approximately 90% of the adult population in the UnitedStates. Active viral replication and infection is kept in check by theimmune system, but, as in cases of CMV, individuals immunocompromised bytransplantation therapies lose the controlling T cell populations, whichpermits viral reactivation. This represents a serious impediment totransplant protocols. EBV may also be involved in tumor promotion in avariety of hematological and non-hematological cancers.

In some embodiments, the patient has an autoimmune disease, in whichenrichment and expansion of regulatory T cells (e.g., CD4+, CD25+,Foxp3+) ex vivo for adoptive transfer to the patient could dampen thedeleterious immune response. Autoimmune diseases that can be treatedinclude systemic lupus erythematosus, rheumatoid arthritis, type Idiabetes, multiple sclerosis, Crohn's disease, ulcerative colitis,psoriasis, myasthenia gravis, Goodpasture's syndrome, Graves' disease,pemphigus vulgaris, Addison's disease, dermatitis herpetiformis, celiacdisease, and Hashimoto's thyroiditis. In some embodiments, the patientis suspected of having an autoimmune disease or immune condition (suchas those described in the preceding sentence), and the evaluation of Tcell responses against a library of paramagnetic nano-aAPCs as describedherein, is useful for identifying or confirming the immune condition.

Thus, in various embodiments the invention involves enrichment andexpansion of antigen-specific T cells, such as cytotoxic T lymphocytes(CTLs), helper T cells, or regulatory T cells. In some embodiments, theinvention involves enrichment and expansion of antigen-specific CTLs.Precursor T cells can be obtained from the patient or from a suitableHLA-matched donor. Precursor T cells can be obtained from a number ofsources, including peripheral blood mononuclear cells (PBMC), bonemarrow, lymph node tissue, spleen tissue, and tumors. In someembodiments, the sample is a PBMC sample from the patient. In someembodiments, the PBMC sample is used to isolate the T cell population ofinterest, such as CD8+, CD4+ or regulatory T cells. In some embodiments,precursor T cells are obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as FICOLL separation. For example, precursor T cells from thecirculating blood of an individual can be obtained by apheresis orleukapheresis. The apheresis product typically contains lymphocytes,including T cells and precursor T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. Leukapheresis is a laboratory procedure in which white bloodcells are separated from a sample of blood.

Cells collected by apheresis can be washed to remove the plasma fractionand to place the cells in an appropriate buffer or media for subsequentprocessing steps. Washing steps can be accomplished by methods known tothose in the art, such as by using a semi-automated “flow-through”centrifuge (for example, the Cobe 2991 cell processor) according to themanufacturer's instructions. After washing, the cells may be resuspendedin a variety of biocompatible buffers, such as, for example, Ca-free,Mg-free PBS. Alternatively, the undesirable components of the apheresissample can be removed and the cells directly re-suspended in a culturemedium.

If desired, precursor T cells can be isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™gradient.

If desired, subpopulations of T cells can be separated from other cellsthat may be present. For example, specific subpopulations of T cells,such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be furtherisolated by positive or negative selection techniques. Other enrichmenttechniques include cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry, e.g., using a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected.

In certain embodiments, leukocytes are collected by leukapheresis, andare subsequently enriched for CD8+ T cells using known processes, suchas magnetic enrichment columns that are commercially available. TheCD8-enriched cells are then enriched for antigen-specific T cells usingmagnetic enrichment with the aAPC reagent. In various embodiments, atleast about 10⁵, or at least about 10⁶, or at least about 10⁷CD8-enriched cells are isolated for antigen-specific T cell enrichment.

In various embodiments, the sample comprising the immune cells (e.g.,CD8+ T cells) is contacted with an artificial Antigen Presenting Cell(aAPC) having magnetic properties. Paramagnetic materials have a small,positive susceptibility to magnetic fields. These materials areattracted by a magnetic field and the material does not retain themagnetic properties when the external field is removed. Exemplaryparamagnetic materials include, without limitation, magnesium,molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beadssuitable for magnetic enrichment are commercially available (DYNABEADS™,MACS MICROBEADS™, Miltenyi Biotec). In some embodiments, the aAPCparticle is an iron dextran bead (e.g., dextran-coated iron-oxide bead).

In certain embodiments, the aAPCs contain at least two ligands, anantigen presenting complex (peptide-MHC), and a lymphocyte activatingligand.

Antigen presenting complexes comprise an antigen binding cleft, whichharbors an antigen for presentation to a T cell or T cell precursor.Antigen presenting complexes can be, for example, MHC class I or classII molecules, and can be linked or tethered to provide dimeric ormultimeric MHC. In some embodiments, the MHC are monomeric, but theirclose association on the nano-particle is sufficient for avidity andactivation. In some embodiments, the MHC are dimeric. Dimeric MHC classI constructs can be constructed by fusion to immunoglobulin heavy chainsequences, which are then associated through one or more disulfide bonds(and with associated light chains). In some embodiments, the signal 1complex is a non-classical MHC-like molecule such as member of the CD1family (e.g., CD1a, CD1b, CD1c, CD1d, and CD1e). MHC multimers can becreated by direct tethering through peptide or chemical linkers, or canbe multimeric via association with streptavidin through biotin moieties.In some embodiments, the antigen presenting complexes are MHC class I orMHC class II molecular complexes involving fusions with immunoglobulinsequences, which are extremely stable and easy to produce, based on thestability and secretion efficiency provided by the immunoglobulinbackbone.

MHC class I molecular complexes having immunoglobulin sequences aredescribed in U.S. Pat. No. 6,268,411, which is hereby incorporated byreference in its entirety. These MHC class I molecular complexes may beformed in a conformationally intact fashion at the ends ofimmunoglobulin heavy chains. MHC class I molecular complexes to whichantigenic peptides are bound can stably bind to antigen-specificlymphocyte receptors (e.g., T cell receptors). In various embodiments,the immunoglobulin heavy chain sequence is not full length, butcomprises an Ig hinge region, and one or more of CH1, CH2, and/or CH3domains. The Ig sequence may or may not comprise a variable region, butwhere variable region sequences are present, the variable region may befull or partial. The complex may further comprise immunoglobulin lightchains.

Exemplary MHC class I molecular complexes comprise at least two fusionproteins. A first fusion protein comprises a first MHC class I a chainand a first immunoglobulin heavy chain (or portion thereof comprisingthe hinge region), and a second fusion protein comprises a second MHCclass I α chain and a second immunoglobulin heavy chain (or portionthereof comprising the hinge region). The first and secondimmunoglobulin heavy chains associate to form the MHC class I molecularcomplex, which comprises two MHC class I peptide-binding clefts. Theimmunoglobulin heavy chain can be the heavy chain of an IgM, IgD, IgG1,IgG3, IgG2β, IgG2α, IgG4, IgE, or IgA. In some embodiments, an IgG heavychain is used to form MHC class I molecular complexes. If multivalentMHC class I molecular complexes are desired, IgM or IgA heavy chains canbe used to provide pentavalent or tetravalent molecules, respectively.

Exemplary class I molecules include HLA-A, HLA-B, HLA-C, HLA-E, andthese may be employed individually or in any combination. In someembodiments, the antigen presenting complex is an HLA-A2 ligand.

Exemplary MHC class II molecular complexes are described in U.S. Pat.Nos. 6,458,354, 6,015,884, 6,140,113, and U.S. Pat. No. 6,448,071, whichare hereby incorporated by reference in their entireties. MHC class IImolecular complexes comprise at least four fusion proteins. Two firstfusion proteins comprise (i) an immunoglobulin heavy chain (or portionthereof comprising the hinge region) and (ii) an extracellular domain ofan MHC class IIβ chain. Two second fusion proteins comprise (i) animmunoglobulin κ or λ light chain (or portion thereof) and (ii) anextracellular domain of an MHC class IIα chain. The two first and thetwo second fusion proteins associate to form the MHC class II molecularcomplex. The extracellular domain of the MHC class IIβ chain of eachfirst fusion protein and the extracellular domain of the MHC class IIαchain of each second fusion protein form an MHC class II peptide bindingcleft.

The immunoglobulin heavy chain can be the heavy chain of an IgM, IgD,IgG3, IgG1, IgG2β, IgG2α, IgG4, IgE, or IgA. In some embodiments, anIgG1 heavy chain is used to form divalent molecular complexes comprisingtwo antigen binding clefts. Optionally, a variable region of the heavychain can be included. IgM or IgA heavy chains can be used to providepentavalent or tetravalent molecular complexes, respectively.

Fusion proteins of an MHC class II molecular complex can comprise apeptide linker inserted between an immunoglobulin chain and anextracellular domain of an MHC class II polypeptide. The length of thelinker sequence can vary, depending upon the flexibility required toregulate the degree of antigen binding and receptor cross linking.

Immunoglobulin sequences in some embodiments are humanized monoclonalantibody sequences.

Signal 2 is generally a T cell affecting molecule, that is, a moleculethat has a biological effect on a precursor T cell or on anantigen-specific T cell. Such biological effects include, for example,differentiation of a precursor T cell into a CTL, helper T cell (e.g.,Th1, Th2), or regulatory T cell; and/or proliferation of T cells. Thus,T cell affecting molecules include T cell costimulatory molecules,adhesion molecules, T cell growth factors, and regulatory T cell inducermolecules. In some embodiments, an aAPC comprises at least one suchligand; optionally, an aAPC comprises at least two, three, or four suchligands.

In certain embodiments, signal 2 is a T cell costimulatory molecule. Tcell costimulatory molecules contribute to the activation ofantigen-specific T cells. Such molecules include, but are not limitedto, molecules that specifically bind to CD28 (including antibodies),CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134(OX-40L), B7h (B7RP-1), CD40, LIGHT, antibodies that specifically bindto HVEM, antibodies that specifically bind to CD40L, antibodies thatspecifically bind to OX40, and antibodies that specifically bind to4-1BB. In some embodiments, the costimulatory molecule (signal 2) is anantibody (e.g., a monoclonal antibody) or portion thereof, such asF(ab′)₂, Fab, scFv, or single chain antibody, or other antigen bindingfragment. In some embodiments, the antibody is a humanized monoclonalantibody or portion thereof having antigen-binding activity, or is afully human antibody or portion thereof having antigen-binding activity.

Adhesion molecules useful for nano-aAPC can be used to mediate adhesionof the nano-aAPC to a T cell or to a T cell precursor. Useful adhesionmolecules include, for example, ICAM-1 and LFA-3.

In some embodiments, signal 1 is provided by peptide-HLA-A2 complexes,and signal 2 is provided by B7.1-Ig or anti-CD28. An exemplary anti-CD28monoclonal antibody is 9.3 mAb (Tan et al., J. Exp. Med. 1993 177:165),which may be humanized in certain embodiments and/or conjugated to thebead as a fully intact antibody or an antigen-binding fragment thereof.

Some embodiments employ T cell growth factors, which affectproliferation and/or differentiation of T cells. Examples of T cellgrowth factors include cytokines (e.g., interleukins, interferons) andsuperantigens. If desired, cytokines can be present in molecularcomplexes comprising fusion proteins, or can be encapsulated by theaAPC. Particularly useful cytokines include IL-2, IL-4, IL-7, IL-10,IL-12, IL-15, IL-21 gamma interferon, and CXCL10. Optionally, cytokinesare provided solely by media components during expansion steps.

The nanoparticles can be made of any material, and materials can beappropriately selected for the desired magnetic property, and maycomprise, for example, metals such as iron, nickel, cobalt, or alloy ofrare earth metal. Paramagnetic materials also include magnesium,molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beadssuitable for enrichment of materials (including cells) are commerciallyavailable, and include iron dextran beads, such as dextran-coated ironoxide beads. In aspects of the invention where magnetic properties arenot required, nanoparticles can also be made of nonmetal or organic(e.g., polymeric) materials such as cellulose, ceramics, glass, nylon,polystyrene, rubber, plastic, or latex. In exemplary material forpreparation of nanoparticles is poly(lactic-co-glycolic acid) (PLGA) andcopolymers thereof, which may be employed in connection with theseembodiments. Other materials including polymers and co-polymers that maybe employed include those described in PCT/US2014/25889, which is herebyincorporated by reference in its entirety.

In some embodiments, the magnetic particles are biocompatible. This isparticularly important in embodiments where the aAPC will be deliveredto the patient in association with the enriched and expanded cells. Forexample, in some embodiments, the magnetic particles are biocompatibleiron dextran paramagnetic beads.

In various embodiments, the particle has a size (e.g., average diameter)within about 10 to about 500 nm, or within about 20 to about 200 nm.Especially in embodiments where aAPC will be delivered to patients,microscale aAPC are too large to be carried by lymphatics, and wheninjected subcutaneously remain at the injection site. When injectedintravenously, they lodge in capillary beds. In fact, the poortrafficking of microscale beads has precluded the study of where aAPCshould traffic for optimal immunotherapy. Trafficking andbiodistribution of nano-aAPC is likely to be more efficient thanmicroscale aAPC. For example, two potential sites where an aAPC might bemost effective are the lymph node, where naive and memory T cellsreside, and the tumor itself. Nanoparticles of about 50 to about 200 nmdiameter can be taken up by lymphatics and transported to the lymphnodes, thus gaining access to a larger pool of T cells. As described inPCT/US2014/25889, which is hereby incorporated by reference,subcutaneous injection of nano-aAPCs resulted in less tumor growth thancontrols or intravenously injected beads.

In some embodiments, re-enrichment of antigen-specific T cells usingnano-sized aAPC, just prior to infusion of the T cells into the patient,will avoid blockage of veins and arteries, for example, which could bean effect if micro-sized aAPCs were infused into the patient along withcells.

Receptor-ligand interactions at the cell-nanoparticle interface are notwell understood. However, nanoparticle binding and cellular activationare sensitive to membrane spatial organization, which is particularlyimportant during T cell activation, and magnetic fields can be used tomanipulate cluster-bound nanoparticles to enhance activation. SeeWO/2014/160132. For example, T cell activation induces a state ofpersistently enhanced nanoscale TCR clustering and nanoparticles aresensitive to this clustering in a way that larger particles are not. SeeWO/2014/160132.

Furthermore, nanoparticle interactions with TCR clusters can beexploited to enhance receptor triggering. T cell activation is mediatedby aggregation of signaling proteins, with “signaling clusters” hundredsof nanometers across, initially forming at the periphery of the Tcell-APC contact site and migrating inward. As described herein, anexternal magnetic field can be used to enrich antigen-specific T cells(including rare naïve cells) and to drive aggregation of magneticnano-aAPC bound to TCR, resulting in aggregation of TCR clusters andenhanced activation of naïve T cells. Magnetic fields can exertappropriately strong forces on paramagnetic particles, but are otherwisebiologically inert, making them a powerful tool to control particlebehavior. T cells bound to paramagnetic nano-aAPC are activated in thepresence of an externally applied magnetic field. Nano-aAPC arethemselves magnetized, and attracted to both the field source and tonearby nanoparticles in the field, inducing bead and thus TCRaggregation to boost aAPC-mediated activation. See WO/2014/150132.

Nano-aAPCs bind more TCR on and induce greater activation of previouslyactivated compared to naive T cells. In addition, application of anexternal magnetic field induces nano-aAPC aggregation on naive cells,enhancing T cells proliferation both in vitro and following adoptivetransfer in vivo. Importantly, in a melanoma adoptive immunotherapymodel, T cells activated by nano-aAPC in a magnetic field mediate tumorrejection. Thus, the use of applied magnetic fields permits activationof naive T cell populations, which otherwise are poorly responsive tostimulation. This is an important feature of immunotherapy as naive Tcells have been shown to be more effective than more differentiatedsubtypes for cancer immunotherapy, with higher proliferative capacityand greater ability to generate strong, long-term T cell responses.Thus, nano-aAPC can used for magnetic field enhanced activation of Tcells to increase the yield and activity of antigen-specific T cellsexpanded from naive precursors, improving cellular therapy for example,patients with infectious diseases, cancer, or autoimmune diseases, or toprovide prophylactic protection to immunosuppressed patients.

Molecules can be directly attached to nanoparticles by adsorption or bydirect chemical bonding, including covalent bonding. See, Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, New York, 1996. A moleculeitself can be directly activated with a variety of chemicalfunctionalities, including nucleophilic groups, leaving groups, orelectrophilic groups. Activating functional groups include alkyl andacyl halides, amines, sulfhydryls, aldehydes, unsaturated bonds,hydrazides, isocyanates, isothiocyanates, ketones, and other groupsknown to activate for chemical bonding. Alternatively, a molecule can bebound to a nanoparticle through the use of a small molecule-couplingreagent. Non-limiting examples of coupling reagents includecarbodiimides, maleimides, n-hydroxysuccinimide esters,bischloroethylamines, bifunctional aldehydes such as glutaraldehyde,anyhydrides and the like. In other embodiments, a molecule can becoupled to a nanoparticle through affinity binding such as abiotin-streptavidin linkage or coupling, as is well known in the art.For example, streptavidin can be bound to a nanoparticle by covalent ornon-covalent attachment, and a biotinylated molecule can be synthesizedusing methods that are well known in the art.

If covalent binding to a nanoparticle is contemplated, the support canbe coated with a polymer that contains one or more chemical moieties orfunctional groups that are available for covalent attachment to asuitable reactant, typically through a linker. For example, amino acidpolymers can have groups, such as the ε-amino group of lysine, availableto couple a molecule covalently via appropriate linkers. This disclosurealso contemplates placing a second coating on a nanoparticle to providefor these functional groups.

Activation chemistries can be used to allow the specific, stableattachment of molecules to the surface of nanoparticles. There arenumerous methods that can be used to attach proteins to functionalgroups. For example, the common cross-linker glutaraldehyde can be usedto attach protein amine groups to an aminated nanoparticle surface in atwo-step process. The resultant linkage is hydrolytically stable. Othermethods include use of cross-linkers containing n-hydrosuccinimido (NHS)esters which react with amines on proteins, cross-linkers containingactive halogens that react with amine-, sulfhydryl-, orhistidine-containing proteins, cross-linkers containing epoxides thatreact with amines or sulfhydryl groups, conjugation between maleimidegroups and sulfhydryl groups, and the formation of protein aldehydegroups by periodate oxidation of pendant sugar moieties followed byreductive amination.

The ratio of particular ligands on the same nanoparticle can be variedto increase the effectiveness of the nanoparticle in antigen orcostimulatory ligand presentation. For example, nanoparticles can becoupled with HLA-A2-Ig and anti-CD28 at a variety of ratios, such asabout 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 5:1,about 3:1, about 2:1, about 1:1, about 0.5:1, about 0.3:1; about 0.2:1,about 0.1:1, or about 0.03:1. The total amount of protein coupled to thesupports may be, for example, about 250 mg/ml, about 200 mg/ml, about150 mg/ml, about 100 mg/ml, or about 50 mg/ml of particles. Becauseeffector functions such as cytokine release and growth may havediffering requirements for Signal 1 versus Signal 2 than T cellactivation and differentiation, these functions can be determinedseparately.

The configuration of nanoparticles can vary from being irregular inshape to being spherical and/or from having an uneven or irregularsurface to having a smooth surface. Non-spherical aAPCs are described inWO 2013/086500, which is hereby incorporated by reference in itsentirety.

In some embodiments, from about 3 to about 10 different antigens arepresented by the antigen presenting complexes in the population ofparamagnetic particles.

The aAPCs present antigen to T cells and thus can be used to both enrichfor and expand antigen-specific T cells, including from naïve T cells.The peptide antigens will be selected based on the desired therapy, forexample, cancer, type of cancer, infectious disease, etc. In someembodiments, the method is conducted to treat a cancer patient, andneoantigens specific to the patient are identified, and synthesized forloading aAPCs. In some embodiments, between three and ten neoantigensare identified through genetic analysis of the tumor (e.g., nucleic acidsequencing), followed by predictive bioinformatics. As shown herein,several antigens can be employed together (on separate aAPCs), with noloss of functionality in the method. In some embodiments, the antigensare natural, non-mutated, cancer antigens, of which many are known. Thisprocess for identifying antigens on a personalized basis is described ingreater detail below.

A variety of antigens can be bound to antigen presenting complexes. Thenature of the antigens depends on the type of antigen presenting complexthat is used. For example, peptide antigens can be bound to MHC class Iand class II peptide binding clefts. Non-classical MHC-like moleculescan be used to present non-peptide antigens such as phospholipids,complex carbohydrates, and the like (e.g., bacterial membrane componentssuch as mycolic acid and lipoarabinomannan). Any peptide capable ofinducing an immune response can be bound to an antigen presentingcomplex. Antigenic peptides include tumor-associated antigens,autoantigens, alloantigens, and antigens of infectious agents.

“Tumor-associated antigens” include unique tumor antigens expressedexclusively by the tumor from which they are derived, shared tumorantigens expressed in many tumors but not in normal adult tissues(oncofetal antigens), and tissue-specific antigens expressed also by thenormal tissue from which the tumor arose. Tumor associated antigens canbe, for example, embryonic antigens, antigens with abnormalpost-translational modifications, differentiation antigens, products ofmutated oncogenes or tumor suppressors, fusion proteins, or oncoviralproteins.

A variety of tumor-associated antigens are known in the art, and many ofthese are commercially available. Oncofetal and embryonic antigensinclude carcinoembryonic antigen and alpha-fetoprotein (usually onlyhighly expressed in developing embryos but frequently highly expressedby tumors of the liver and colon, respectively), MAGE-1 and MAGE-3(expressed in melanoma, breast cancer, and glioma), placental alkalinephosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 andCA-19 (expressed in gastrointestinal, hepatic, and gynecologicaltumors), TAG-72 (expressed in colorectal tumors), epithelialglycoprotein 2 (expressed in many carcinomas), pancreatic oncofetalantigen, 5T4 (expressed in gastriccarcinoma), alphafetoprotein receptor(expressed in multiple tumor types, particularly mammary tumors), andM2A (expressed in germ cell neoplasia).

Tumor-associated differentiation antigens include tyrosinase (expressedin melanoma) and particular surface immunoglobulins (expressed inlymphomas).

Mutated oncogene or tumor-suppressor gene products include Ras and p53,both of which are expressed in many tumor types, Her-2/neu (expressed inbreast and gynecological cancers), EGF-R, estrogen receptor,progesterone receptor, retinoblastoma gene product, myc (associated withlung cancer), ras, p53, nonmutant associated with breast tumors, MAGE-1,and MAGE-3 (associated with melanoma, lung, and other cancers). Fusionproteins include BCR-ABL, which is expressed in chromic myeloidleukemia. Oncoviral proteins include HPV type 16, E6, and E7, which arefound in cervical carcinoma.

Tissue-specific antigens include melanotransferrin and MUC1 (expressedin pancreatic and breast cancers); CD10 (previously known as commonacute lymphoblastic leukemia antigen, or CALLA) or surfaceimmunoglobulin (expressed in B cell leukemias and lymphomas); the achain of the IL-2 receptor, T cell receptor, CD45R, CD4+/CD8+ (expressedin T cell leukemias and lymphomas); prostate specific antigen andprostatic acid-phosphatase (expressed in prostate carcinoma); GP 100,MelanA/Mart-1, tyrosinase, gp75/brown, BAGE, and S-100 (expressed inmelanoma); cytokeratins (expressed in various carcinomas); and CD19,CD20, and CD37 (expressed in lymphoma).

Tumor-associated antigens also include altered glycolipid andglycoprotein antigens, such as neuraminic acid-containingglycosphingolipids (e.g., GM2 and GD2, expressed in melanomas and somebrain tumors); blood group antigens, particularly T and sialylated Tnantigens, which can be aberrantly expressed in carcinomas; and mucins,such as CA-125 and CA-19-9 (expressed on ovarian carcinomas) or theunderglycosylated MUC-1 (expressed on breast and pancreatic carcinomas).

“Antigens of infectious agents” include components of protozoa,bacteria, fungi (both unicellular and multicellular), viruses, prions,intracellular parasites, helminths, and other infectious agents that caninduce an immune response.

Bacterial antigens include antigens of gram-positive cocci, grampositive bacilli, gram-negative bacteria, anaerobic bacteria, such asorganisms of the families Actinomycetaceae, Bacillaceae, Bartonellaceae,Bordetellae, Captophagaceae, Corynebacteriaceae, Enterobacteriaceae,Legionellaceae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae,Pasteurellaceae, Pseudomonadaceae, Spirochaetaceae, Vibrionaceae andorganisms of the genera Acinetobacter, Brucella, Campylobacter,Erysipelothrix, Ewingella, Francisella, Gardnerella, Helicobacter,Levinea, Listeria, Streptobacillus and Tropheryma.

Antigens of protozoan infectious agents include antigens of malarialplasmodia, Leishmania species, Trypanosoma species and Schistosomaspecies.

Fungal antigens include antigens of Aspergillus, Blastomyces, Candida,Coccidioides, Cryptococcus, Histoplasma, Paracoccicioides, Sporothrix,organisms of the order Mucorales, organisms inducing choromycosis andmycetoma and organisms of the genera Trichophyton, Microsporum,Epidermophyton, and Malassezia.

Viral peptide antigens include, but are not limited to, those ofadenovirus, herpes simplex virus, papilloma virus, respiratory syncytialvirus, poxviruses, HIV, influenza viruses, and CMV. Particularly usefulviral peptide antigens include HIV proteins such as HIV gag proteins(including, but not limited to, membrane anchoring (MA) protein, corecapsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase,influenza virus matrix (M) protein and influenza virus nucleocapsid (NP)protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein(HBcAg), hepatitis e protein (HBeAg), hepatitis B DNA polymerase,hepatitis C antigens, and the like.

Antigens, including antigenic peptides, can be bound to an antigenbinding cleft of an antigen presenting complex either actively orpassively, as described in U.S. Pat. No. 6,268,411, which is herebyincorporated by reference in its entirety. Optionally, an antigenicpeptide can be covalently bound to a peptide binding cleft.

If desired, a peptide tether can be used to link an antigenic peptide toa peptide binding cleft. For example, crystallographic analyses ofmultiple class I WIC molecules indicate that the amino terminus of β2Mis very close, approximately 20.5 Angstroms away, from the carboxylterminus of an antigenic peptide resident in the MHC peptide bindingcleft. Thus, using a relatively short linker sequence, approximately 13amino acids in length, one can tether a peptide to the amino terminus ofβ2M. If the sequence is appropriate, that peptide will bind to the MHCbinding groove (see U.S. Pat. No. 6,268,411).

Antigen-specific T cells which are bound to the aAPCs can be separatedfrom cells which are not bound using magnetic enrichment, or other cellsorting or capture technique. Other processes that can be used for thispurpose include flow cytometry and other chromatographic means (e.g.,involving immobilization of the antigen-presenting complex or otherligand described herein). In one embodiment antigen-specific T cells areisolated (or enriched) by incubation with beads, for example,antigen-presenting complex/anti-CD28-conjugated paramagnetic beads (suchas DYNABEADS®), for a time period sufficient for positive selection ofthe desired antigen-specific T cells.

In some embodiments, a population of T cells can be substantiallydepleted of previously active T cells using, e.g., an antibody to CD44,leaving a population enriched for naïve T cells. Binding nano-aAPCs tothis population would not substantially activate the naïve T cells, butwould permit their purification.

In still other embodiments, ligands that target NK cells, NKT cells, orB cells (or other immune effector cells), can be incorporated into aparamagnetic nanoparticle, and used to magnetically enrich for thesecell populations, optionally with expansion in culture as describedbelow. Additional immune effector cell ligands are described inPCT/US2014/25889, which is hereby incorporated by reference in itsentirety.

Without wishing to be bound by theory, removal of unwanted cells mayreduce competition for cytokines and growth signals, remove suppressivecells, or may simply provide more physical space for expansion of thecells of interest.

Enriched T cells are then expanded in culture within the proximity of amagnet to produce a magnetic field, which enhances T cell receptorclustering of aAPC bound cells. Cultures can be stimulated for variableamounts of time (e.g., about 0.5, 2, 6, 12, 36, 48, or 72 hours as wellas continuous stimulation) with nano-aAPC. The effect of stimulationtime in highly enriched antigen-specific T cell cultures can beassessed. Antigen-specific T cell can be placed back in culture andanalyzed for cell growth, proliferation rates, various effectorfunctions, and the like, as is known in the art. Such conditions mayvary depending on the antigen-specific T cell response desired. In someembodiments, T cells are expanded in culture from about 2 days to about3 weeks, or in some embodiments, about 5 days to about 2 weeks, or about5 days to about 10 days. In some embodiments, the T cells are expandedin culture for about 1 week, after which time a second enrichment andexpansion step is optionally performed. In some embodiments, 2, 3, 4, or5 enrichment and expansion rounds are performed.

After the one or more rounds of enrichment and expansion, theantigen-specific T cell component of the sample will be at least about1% of the cells, or in some embodiments, at least about 5%, at leastabout 10%, at least about 15%, or at least about 20%, or at least about25% of the cells in the sample. Further, these T cells generally displayan activated state. From the original sample isolated from the patient,the antigen-specific T cells in various embodiments are expanded fromabout 100-fold to about 10,000 fold, such as at least about 1000-fold,at least about 2000-fold, at least about 3,000 fold, at least about4,000-fold, or at least about 5,000-fold in various embodiments. Afterthe one or more rounds of enrichment and expansion, at least about 10⁶,or at least about 10⁷, or at least about 10⁸, or at least about 10⁹antigen-specific T cells are obtained.

The effect of nano-aAPC on expansion, activation and differentiation ofT cell precursors can be assayed in any number of ways known to those ofskill in the art. A rapid determination of function can be achievedusing a proliferation assay, by determining the increase of CTL, helperT cells, or regulatory T cells in a culture by detecting markersspecific to each type of T cell. Such markers are known in the art. CTLcan be detected by assaying for cytokine production or for cytolyticactivity using chromium release assays.

In addition to generating antigen-specific T cells with appropriateeffector functions, another parameter for antigen-specific T cellefficacy is expression of homing receptors that allow the T cells totraffic to sites of pathology (Sallusto et al., Nature 401, 708-12,1999; Lanzavecchia & Sallusto, Science 290, 92-97, 2000).

For example, effector CTL efficacy has been linked to the followingphenotype of homing receptors, CD62L+, CD45RO+, and CCR7−. Thus, anano-aAPC-induced and/or expanded CTL population can be characterizedfor expression of these homing receptors. Homing receptor expression isa complex trait linked to initial stimulation conditions. Presumably,this is controlled both by the co-stimulatory complexes as well ascytokine milieu. One important cytokine that has been implicated isIL-12 (Salio et al., 2001). As discussed below, nano-aAPC offer thepotential to vary individually separate components (e.g., T celleffector molecules and antigen presenting complexes) to optimizebiological outcome parameters. Optionally, cytokines such as IL-12 canbe included in the initial induction cultures to affect homing receptorprofiles in an antigen-specific T cell population.

Optionally, a cell population comprising antigen-specific T cells cancontinue to be incubated with either the same nano-aAPC or a secondnano-aAPC for a period of time sufficient to form a second cellpopulation comprising an increased number of antigen-specific T cellsrelative to the number of antigen-specific T cells in the first cellpopulation. Typically, such incubations are carried out for 3-21 days,preferably 7-10 days.

Suitable incubation conditions (culture medium, temperature, etc.)include those used to culture T cells or T cell precursors, as well asthose known in the art for inducing formation of antigen-specific Tcells using DC or artificial antigen presenting cells. See, e.g.,Latouche & Sadelain, Nature Biotechnol. 18, 405-09, April 2000; Levineet al., J. Immunol. 159, 5921-30, 1997; Maus et al., Nature Biotechnol.20, 143-48, February 2002. See also the specific examples, below.

To assess the magnitude of a proliferative signal, antigen-specific Tcell populations can be labeled with CFSE and analyzed for the rate andnumber of cell divisions. T cells can be labeled with CFSE after one-tworounds of stimulation with nano-aAPC to which an antigen is bound. Atthat point, antigen-specific T cells should represent 2-10% of the totalcell population. The antigen-specific T cells can be detected usingantigen-specific staining so that the rate and number of divisions ofantigen-specific T cells can be followed by CFSE loss. At varying times(for example, 12, 24, 36, 48, and 72 hours) after stimulation, the cellscan be analyzed for both antigen presenting complex staining and CFSE.Stimulation with nano-aAPC to which an antigen has not been bound can beused to determine baseline levels of proliferation. Optionally,proliferation can be detected by monitoring incorporation of3H-thymidine, as is known in the art.

Antigen-specific T cells obtained using nano-aAPC, can be administeredto patients by any appropriate routes, including intravenousadministration, intra-arterial administration, subcutaneousadministration, intradermal administration, intralymphaticadministration, and intratumoral administration. Patients include bothhuman and veterinary patients.

Antigen-specific regulatory T cells can be used to achieve animmunosuppressive effect, for example, to treat or prevent graft versushost disease in transplant patients, or to treat or prevent autoimmunediseases, such as those listed above, or allergies. Uses of regulatory Tcells are disclosed, for example, in US 2003/0049696, US 2002/0090724,US 2002/0090357, US 2002/0034500, and US 2003/0064067, which are herebyincorporated by reference in their entireties.

Antigen-specific T cells prepared according to these methods can beadministered to patients in doses ranging from about 5-10×10⁶ CTL/kg ofbody weight (˜7×10⁸ CTL/treatment) up to about 3.3×10⁹ CTL/kg of bodyweight (˜6×10⁹ CTL/treatment) (Walter et al., New England Journal ofMedicine 333, 1038-44, 1995; Yee et al., J Exp Med 192, 1637-44, 2000).In other embodiments, patients can receive about 10³, about 5×10³, about10⁴, about 5×10⁴, about 10⁵, about 5×10⁵, about 10⁶, about 5×10⁶, about10⁷, about 5×10⁷, about 10⁸, about 5×10⁸, about 10⁹, about 5×10⁹, orabout 10¹⁰ cells per dose administered intravenously. In still otherembodiments, patients can receive intranodal injections of, e.g., about8×10⁶ or about 12×10⁶ cells in a 200 μl bolus. Doses of nano-APC thatare administered with cells include about 10³, about 5×10³, about 10⁴,about 5×10⁴, about 10⁵, about 5×10⁵, about 10⁶, about 5×10⁶, about 10⁷,about 5×10⁷, about 10⁸, about 5×10⁸, about 10⁹, about 5×10⁹, or about10¹⁰ nano-aAPC per dose.

In an exemplary embodiment, the enrichment and expansion process isperformed repeatedly on the same sample derived from a patient. Apopulation of T cells is enriched and activated on Day 0, followed by asuitable period of time (e.g., about 3-20 days) in culture.Subsequently, nano-aAPC can be used to again enrich and expand againstthe antigen of interest, further increasing population purity andproviding additional stimulus for further T cell expansion. The mixtureof nano-aAPC and enriched T cells may subsequently again be cultured invitro for an appropriate period of time, or immediately re-infused intoa patient for further expansion and therapeutic effect in vivo.Enrichment and expansion can be repeated any number of times until thedesired expansion is achieved.

In some embodiments, a cocktail of nano-aAPC, each against a differentantigen, can be used at once to enrich and expand antigen T cellsagainst multiple antigens simultaneously. In this embodiment, a numberof different nano-aAPC batches, each bearing a different MHC-peptide,would be combined and used to simultaneously enrich T cells against eachof the antigens of interest. In some embodiments, from about 3 to about10 different antigens are presented by the antigen presenting complexes.The resulting T cell pool would be enriched and activated against eachof these antigens, and responses against multiple antigens could thus becultured simultaneously. These antigens could be related to a singletherapeutic intervention; for example, multiple antigens present on asingle tumor.

In some embodiments, the patient receives immunotherapy with one or morecheckpoint inhibitors, prior to receiving the antigen-specific T cellsby adoptive transfer, or prior to direct administration of aAPCs bearingneoantigens identified in vitro through genetic analysis of thepatient's tumor. In various embodiments, the checkpoint inhibitor(s)target one or more of CTLA-4 or PD-1/PD-L1, which may include antibodiesagainst such targets, such as monoclonal antibodies, or portionsthereof, or humanized or fully human versions thereof. In someembodiments, the checkpoint inhibitor therapy comprises ipilimumab orKeytruda (pembrolizumab).

In some embodiments, the patient receives about 1 to 5 rounds ofadoptive immunotherapy (e.g., one, two, three, four or five rounds). Insome embodiments, each administration of adoptive immunotherapy isconducted simultaneously with, or after (e.g., from about 1 day to about1 week after), a round of checkpoint inhibitor therapy. In someembodiments, adoptive immunotherapy is provided about 1 day, about 2days, or about 3 days after checkpoint inhibitor therapy.

In still other embodiments, adoptive transfer or direct infusion ofnano-aAPCs to the patient comprises, as a ligand on the bead, a ligandthat targets one or more of CTLA-4 or PD-1/PD-L1. In these embodiments,the method can avoid certain side effects of administering solublecheckpoint inhibitor therapy.

Methods for Personalized Therapy

In some aspects, the invention provides methods for personalized cancerimmunotherapy. The methods are accomplished using the aAPCs to identifyantigens to which the patient will respond, followed by administrationof the appropriate peptide-loaded aAPC to the patient, or followed byenrichment and expansion of the antigen specific T cells ex vivo.

Genome-wide sequencing has dramatically altered our understanding ofcancer biology. Sequencing of cancers has yielded important dataregarding the molecular processes involved in the development of manyhuman cancers. Driving mutations have been identified in key genesinvolved in pathways regulating three main cellular processes (1) cellfate, (2) cell survival and (3) genome maintenance. Vogelstein et al.,Science 339, 1546-58 (2013).

Genome-wide sequencing also has the potential to revolutionize ourapproach to cancer immunotherapy. Sequencing data can provideinformation about both shared as well as personalized targets for cancerimmunotherapy. In principle, mutant proteins are foreign to the immunesystem and are putative tumor-specific antigens. Indeed, sequencingefforts have defined hundred if not thousands of potentially relevantimmune targets. Limited studies have shown that T cell responses againstthese neo-epitopes can be found in cancer patients or induced by cancervaccines. However, the frequency of such responses against a particularcancer and the extent to which such responses are shared betweenpatients are not well known. One of the main reasons for our limitedunderstanding of tumor-specific immune responses is that currentapproaches for validating potential immunologically relevant targets arecumbersome and time consuming.

Thus, in some aspects, the invention provides a high-throughputplatform-based approach for detection of T cell responses againstneo-antigens in cancer. This approach uses the aAPC platform describedherein for the detection of even low-frequency T cell responses againstcancer antigens. Understanding the frequency and between-personvariability of such responses would have important implications for thedesign of cancer vaccines and personalized cancer immunotherapy.

Although central tolerance abrogates T cell responses againstself-proteins, oncogenic mutations induce neo-epitopes against which Tcell responses can form. Mutation catalogues derived from whole exomesequencing provide a starting point for identifying such neo-epitopes.Using HLA binding prediction algorithms (Srivastava, PLoS One 4, e6094(2009), it has been predicted that each cancer can have up 7-10neo-epitopes. A similar approach estimated hundreds of tumorneo-epitopes. Such algorithms, however, may have low accuracy inpredicting T cell responses, and only 10% of predicted HLA-bindingepitopes are expected to bind in the context of HLA (Lundegaard C,Immunology 130, 309-18 (2010)). Thus, predicted epitopes must bevalidated for the existence of T cell responses against those potentialneo-epitopes.

In certain embodiments, the nano-aAPC system is used to screen forneo-epitopes that induce a T cell response in a variety of cancers, orin a particular patient's cancer. Cancers may be genetically analyzed,for example, by whole exome-sequencing. For example, of a panel of 24advanced adenocarcinomas, an average of about 50 mutations per tumorwere identified. Of approximately 20,000 genes analyzed, 1327 had atleast one mutation, and 148 had two or more mutations. 974 missensemutations were identified, with a small additional number of deletionsand insertions.

A list of candidate peptides can be generated from overlapping nineamino acid windows in mutated proteins. All nine-AA windows that containa mutated amino acid, and 2 non-mutated “controls” from each proteinwill be selected. These candidate peptides will be assessedcomputationally for MHC binding using a consensus of MHC bindingprediction algorithms, including NetMHC and stabilized matrix method(SMM). Nano-aAPC and MHC binding algorithms have been developedprimarily for HLA-A2 allele. The sensitivity cut-off of the consensusprediction can be adjusted until a tractable number of mutationcontaining peptides (˜500) and non-mutated control peptides (˜50) areidentified.

A peptide library is then synthesized. MHC (e.g., A2) bearing aAPC aredeposited in multi well plates and passively loaded with peptide. CD8 Tcells may be isolated from PBMC of both A2 positive healthy donors andA2 positive pancreatic cancers patients (or other cancer or diseasedescribed herein). Subsequently, the isolated T cells are incubated withthe loaded aAPCs in the plates for the enrichment step. Following theincubation, the plates are placed on a magnetic field and thesupernatant containing irrelevant T cells not bound to the aAPCs isremoved. The remaining T cells that are bound to the aAPCs will becultured and allowed to expand for 7 to 21 days. Antigen specificexpansion is assessed by re-stimulation with aAPC and intracellular IFNγfluorescent staining.

In some embodiments, a patient's T cells are screened against an arrayor library of nanoAPCs, and the results are used for diagnostic orprognostic purposes. For example, the number and identity of T cellanti-tumor responses against mutated proteins, overexpressed proteins,and/or other tumor-associated antigens can be used as a biomarker tostratify risk. For example, the number of such T cell responses may beinversely proportionate to the risk of disease progression or risk ofresistance or non-responsiveness to chemotherapy. In other embodiments,the patient's T cells are screened against an array or library ofnano-APCs, and the presence of T cells responses, or the number orintensity of these T cells responses identifies that the patient has asub-clinical tumor, and/or provides an initial understanding of thetumor biology.

In some embodiments, a patient or subject's T cells are screened againstan array or library of paramagnetic aAPCs, each presenting a differentcandidate peptide antigen. This screen can provide a wealth ofinformation concerning the subject or patient's T cell repertoire, andthe results are useful for diagnostic or prognostic purposes. Forexample, the number and identity of T cell anti-tumor responses againstmutated proteins, overexpressed proteins, and/or other tumor-associatedantigens can be used as a biomarker to stratify risk, to monitorefficacy of immunotherapy, or predict outcome of immunotherapytreatment. Further, the number or intensity of such T cell responses maybe inversely proportionate to the risk of disease progression or may bepredictive of resistance or non-responsiveness to chemotherapy. In otherembodiments, a subject's or patient's T cells are screened against anarray or library of nano-APCs each presenting a candidate peptideantigen, and the presence of T cells responses, or the number orintensity of these T cells responses, provides information concerningthe health of the patient, for example, by identifying autoimmunedisease, or identifying that the patient has a sub-clinical tumor. Inthese embodiments, the process not only identifies a potential diseasestate, but provides an initial understanding of the disease biology.

Reagents/Kits

In another aspect of the invention, nano-aAPC can be provided in kitstogether with components for performing the enrichment and expansionprocess. Suitable containers for nano-aAPC include, for example,bottles, vials, syringes, and test tubes. Containers can be formed froma variety of materials, including glass or plastic. A container may havea sterile access port (for example, the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). Optionally, one or more different antigens can bebound to the nano-aAPC or can be supplied separately. Kits may comprise,alternatively or in addition, one or more multiwall plates or cultureplates for T cells. In some embodiments, kits comprise a sealedcontainer comprising aAPCS, a magnet, and optionally test tubes and/orsolution or buffers for performing magnetic enrichment.

A kit can further comprise a second container comprising apharmaceutically acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to an end user, including other buffers, diluents,filters, needles, and syringes.

Kits also may contain reagents for assessing the extent and efficacy ofantigen-specific T cell activation or expansion, such as antibodiesagainst specific marker proteins, MHC class I or class II molecularcomplexes, TCR molecular complexes, anticlonotypic antibodies, and thelike.

A kit can also comprise a package insert containing written instructionsfor methods of inducing antigen-specific T cells, expandingantigen-specific T cells, using nanoaAPC in the kit in variousprotocols. The package insert can be an unapproved draft package insertor can be a package insert approved by the Food and Drug Administration(FDA) or other regulatory body.

EXAMPLES Example 1

Adoptive T cell therapy can mediate durable regression of cancer. Torapidly generate large numbers of functional tumor-specific T cells fromnaïve T cells, we developed an Enrichment+Expansion strategy usingparamagnetic, nanoscale artificial Antigen Presenting Cells, capable ofenriching rare tumor-specific T cells in a magnetic column whilesimultaneously activating them. Enrichment+Expansion resulted in greaterthan 1000-fold expansion of mouse and human tumor-specific T cells, andmice treated with tumor-specific CTL generated by Enrichment+Expansionhad significantly less tumor growth. Streamlining the generation oflarge numbers of tumor-specific T cells in a cost effective,reproducible fashion through Enrichment+Expansion could be a powerfuladdition to autologous tumor immunotherapy protocols.

Adoptive transfer of tumor-specific T cells can mediate durableregression of cancer¹. While pre-existing anti-tumor responses can onlybe cultured from a minority of cancer patients², T cells specific for awide variety of tumor antigens can be generated by stimulation of naiveprecursor cells with tumor antigen³. This culture process relies onautologous antigen presenting cells and feeder cells, which are complexbiologics that must be generated for each individual patient⁴,significantly increasing the cost and complexity of adoptiveimmunotherapy.

Expansion of tumor-specific T cells is further complicated by the rarityof tumor-specific naive precursors, as few as one per million⁵⁻⁷. Togenerate the large numbers of tumor-specific T cells required foreffective therapy⁸⁻¹⁰, lymphocytes are repeatedly stimulated withantigen over many weeks, often followed by T cell selection andsub-cloning¹¹. This labor-intensive process increases both the totalnumber and antigen-specific frequency (or “purity”) of tumor-specific Tcells in the final cell product. Antigen-specific frequency is anindependently important parameter for optimal expansion after transfer,since competition for growth signals from irrelevant, co-transferredcells significantly attenuates homeostatic expansion of anti-tumor Tcells of interest¹²⁻¹⁴.

Thus, there is a need for technologies that can quickly generate largenumbers and high frequencies of tumor-specific T cells from naiveprecursors, without the added expense and complexity of cellular APC orfeeder cells. The invention therefore provides a T cell enrichment andexpansion strategy using nanoscale artificial Antigen Presenting Cells(nano-aAPC). The nano-aAPC exemplified here are paramagneticiron-dextran nanoparticles, 50-100 nm in diameter, functionalized withtwo activating signals delivered by endogenous APC: signal 1, a cognateantigenic peptide presented in the context of MHC that binds the TCR;and signal 2, one of a number of co-stimulatory receptors that modulateT cell responses and promote effective activation (FIG. 1, top).Paramagnetic nano-aAPC are thus capable of both capturing cognate Tcells in a magnetic enrichment column, and inducing antigen-specific Tcell expansion (FIG. 1, bottom).

Enrichment with nano-aAPC is performed by incubating naive, polyclonalmouse CD8+ T lymphocytes with nano-aAPC, passing the cell-particlemixture through a magnetic column, eluting and then culturing themagnet-bound fraction (FIG. 1). To assess efficacy of enrichment, aknown number of Thy1.1+ pmel TCR transgenic T cells specific forDb-gp100 melanoma antigen were mixed at a 1:1000 ratio with Thy1.2+ CD8T cells from B6 mice. After enrichment with pmel gp100-specific aAPC,the frequency of antigen-specific pmel T cells increased more than10-fold from 0.07% before enrichment to 1.17% after enrichment in adose-dependent manner (FIG. 2A). Optimizing the amount of nano-aAPCincubated with T cells increased the enrichment efficiency and resultedin recovery of up 95% of the added pmel T cells (FIG. 2B).

Enrichment of wild-type Db-gp100 cells from endogenous B6 CD8+splenocytes was assessed by staining with soluble MHC pentamer. Db-gp100specific frequency was below detectable levels prior to enrichment, butincreased to 0.30% afterward. The frequency of non-specific Kb-Trp2cells incubated with Db-gp100 particles did not increase (FIG. 2C).

After enrichment, magnet-bound fractions (positive fraction) of enrichedcells and nano-aAPC were eluted and cultured in vitro. To study theeffect of enrichment on subsequent proliferation, the enrichmentprocedure was “undone” in control samples by collecting the negativefraction (CD8+ T cells not bound to nano-aAPC), and adding it back tothe positive fraction (FIG. 3A).

Enrichment significantly enhanced antigen-specific frequency and total Tcell number after expansion. Seven days after enrichment with a Kb-Trp2nano-aAPC, 17.6% of cells expanded from the positive fraction wereKb-Trp2 specific, compared to 1.46% of cells from the negative+positive,not enriched group (FIG. 3B). The enrichment procedure resulted in a 2-3fold increase in total antigen-specific cells, despite greater numbersof total cells in the negative+positive fraction. We hypothesize theincrease in total T cell expansion may be mediated by reducedcompetition for lymphotrophic cytokines.

The Enrichment+Expansion approach was broadly applicable to a variety oftumor and model T cell antigens, including melanoma antigen gp100(Db-gp100), the Kb-restricted ovalbumin antigen SIIN (Kb-SIIN), and thecolon carcinoma antigen Ld-AH1/A5 (Ld-A5) (FIGS. 3D, 3E). Absolutenumbers of antigen-specific cells and frequencies wereantigen-dependent. Kb-SIIN responses consistently resulted in 20%antigen-specific cells after one week, whereas Db-gp100 specificitieswere approximately 5% and Ld-A5 approximately 7.5% of total T cells.

T cell proliferation was estimated from known precursor frequencies forthe antigens of interest (Table 1). Precursor frequencies for CD8responses to foreign antigens range from 10 s-100 s/10⁷,⁷ and areexpected to be at the lower end of this range for the self-antigens suchas Trp2. Precursor frequencies for Db-gp100 have been measured at 10 in10⁷,⁵ and 40-350 in 10⁷ for Kb-SIINF. After one week, 150,000Trp2-specific cells were generated from 10⁷ polyclonal CD8 T cells;thus, we estimate Trp2-specific proliferation is between 100-1000 fold.In comparison, approximately 35,000 Db-gp100 and 150,000 Kb-SIINFspecific T cells were generated from 10⁷ T cells, indicating up to 5,000fold expansion for each antigen. This is comparable to the robustexpansion observed after viral infection in vivo¹⁸.

To validate these estimates, we labeled naive T cell populations withthe proliferation marker dye CFSE, which is diluted in half with everyround of T cell division. Four days following E+E, Kb-Trp2 tetramerbinding T cells had diluted their CFSE below detectable limits.Transgenic pmel T cells stimulated with a moderate dose of nano-aAPCwere used for comparison; these cells showed multiple peaks of CFSEfluorescence, indicating between 2-7 rounds of division. This allowed usto determine that enriched+expanded Trp2-specific T cells had completedmore than 7 rounds of division, consistent with greater than 256-foldexpansion after only four days. Expanded T cells showed a CD62L low CD44high effector memory phenotype, consistent with robust activation andproliferation.

T cell expansion by E+E was compared to expansion using mature, bonemarrow derived dendritic cells pulsed with Trp2 peptide. Stimulation often million naive lymphocytes resulted in 2±0.5×10⁴ Trp2-specific Tcells, with antigen-specific frequencies between 0.5-2.85%,approximately 10-fold lower in number and frequency than that achievedwith E+E. This is consistent with expansion by APC and artificial APC inhumans, where antigen-specific responses after one week of stimulationare frequently not detectable¹⁹.

Simultaneous generation of T cell responses to multiple tumor antigenswould increase the number of anti-tumor T cells generated from a singlenaive T cell population, and reduce the likelihood of tumor immuneescape due to down-regulation of a single antigen²⁰⁻²². We thereforedeveloped a single-step E+E protocol for generating multiple anti-tumorpopulations simultaneously.

Naive lymphocytes were incubated with nano-aAPC bearing Db-gp100,Kb-SIINF, and Kb-Trp2 MHC dimers, each at the standard single-antigendose. One week after “triple” E+E, antigen-specific T cells weredetected by pentamer staining against each antigen of interest (FIG.3F). While the frequency of each population was lower than that found incontrol samples stimulated with only one antigen (FIG. 3G), the totalantigen-specific cells was the same whether isolated individually orsimultaneously (p>0.4 by two-way ANOVA) (FIG. 3G). Thus, the triple E+Eprotocol was as efficient for each tumor specific T cell population asany of the single-antigen controls.

Adoptively transferred tumor-specific T cells compete withco-transferred, non-tumor specific bystander cells for growthsignals¹²⁻¹⁴. However, this effect has not been demonstrated forantigen-specific expansion of T cells that have been previouslyactivated in vitro, as occurs during Enrichment+Expansion.

We thus combined tumor-specific pmel T cells and polyclonal, wild-typeB6 cells in ratios that approximate the antigen specific frequenciesachieved with and without E+E (10% and 1%, respectively). In each group,the total number of pmel T cells administered was the same (10⁵); onlythe amount of bystander T cells differed (10⁶ or 10⁷). The largestnumber and highest frequency of pmel T cells were observed in micereceiving fewer (10⁶) bystander cells (FIG. 4A-B). Approximately5.5±1.5×10⁵ pmel T cells were recovered from the spleen and lymph nodesof these animals (FIG. 4B). Only 1.4±0.7×10⁵ pmel T cells were recoveredfrom animals receiving 10⁷ bystander cells (p<0.05 by two-way ANOVA withTukey post-test). Thus, removal of competition from co-transferred cellsenhanced engraftment and expansion after transfer.

In addition, tumor-specific T cells compete with host cells for growthsignals²⁴, which has motivated the use of host radio- and chemo-basedlymphodepletion prior to adoptive transfer^(25,26). Thus, animalsreceiving 10⁶ or 10⁷ bystander cells were either irradiated with 500 cGygamma radiation 24 hours prior to transfer or left untreated, generatingfour experimental groups. Animals that were not irradiated showed poorengraftment, with less than 0.3×10⁵ pmel T cells recovered in either the10⁶ or 10⁷ bystander group (FIG. 4A-B). Thus, removal of bothtransferred bystander lymphocytes and/or host lymphocytes significantlyincreased the yield of adoptively transferred tumor-specific T cells inthe host.

We next determined that tumor-specific lymphocytes generated byEnrichment+Expansion with nano-aAPC mediated rejection of establishedmelanoma. B16-F10 cells, an aggressive, poorly immunogenic melanomamodel, were implanted subcutaneously into B6 host mice and allowed togrow for eight days until tumors were palpable. In parallel, CD8lymphocytes were isolated from naive B6 donor mice and Enriched+Expandedagainst Db-GP100 and Kb-Trp2 antigens, then transferred into hosts oneday after lymphodepletion.

Animals receiving tumor-specific E+E donor lymphocytes had significantlyless tumor growth than untreated mice, or mice receiving equivalentnumbers of lymphocytes generated against irrelevant Kb-SIINF antigen(FIG. 4C). Eighteen days after tumor injection, mean tumor area foruntreated mice was 130±12 mm², compared to 144±11 mm² for Kb-SIINFtreated mice and 22±9 mm² for Db-gp100/Kb-Trp2 treated mice (p<0.05 byANOVA with Tukey post-test).

All mice in untreated and Kb-SIINF treated groups were sacrificed by day22 due to excessive tumor burden. By comparison, no mice in theDb-gp100/Kb-Trp2 group were sacrificed until day 24, and 2/8 mice had nodetectable tumor 2 months after implantation (p<0.01 by Mantel-Cox).Median survival was significantly greater in the E+E treated group (28days) than the untreated (20 days) or non-cognate treated (20 days)group. Thus, E+E lymphocytes cultured from naive cells for only a weekwere able to delay and in some cases completely reject established B16melanoma.

Enrichment+Expansion by nano-aAPC functionalized with HLA-A2 is alsoeffective at expanding human anti-tumor responses from naivelymphocytes. Human CD8+ lymphocytes were isolated from peripheral bloodmononuclear cells of healthy donors, and E+E was performed withnano-aAPC bearing either NY-ESO1 or MART1 tumor antigens. After oneweek, 44,000±21,000 NY-ESO1 specific cells were generated, representingapproximately 1000-fold precursor expansion (Table 1, FIG. 5). For MART1responses, 83,000±37,000 were generated in one week; this representsapproximately 100-fold expansion, reflecting the high precursorfrequency of MART1 responses found even in healthy donors 27 (Table 1,FIG. 5). Thus Enrichment+Expansion is not limited to murine T cells, butis also a robust approach for the expansion of naïve, low frequency,anti-tumor human CTL.

Wide-spread application of adoptive immunotherapy for cancer is limitedby the availability of cost-effective and convenient sources oftumor-specific cells. Here, we developed a streamlined technology forquickly expanding large numbers of high frequency tumor-specificlymphocytes from naive cells, with more than 1000-fold expansion in oneweek. We further demonstrate that removing irrelevant bystander cells byenrichment confers a significant survival and proliferation advantage totumor-specific T cells both during in vitro culture and after adoptivetransfer in vivo.

While antigen-specific T cells can be enriched using MHC tetramers afterT cell expansion²⁸⁻³⁰, our platform simplifies this process by allowingenrichment and expansion to be performed with a single reagent.Furthermore, cross-linking of TCR by multimeric MHC in the absence ofco-stimulation can induce T cell apoptosis or anergy³¹⁻³³, with deletionof up to one-half of antigen-specific cells after tetramer engagement.Thus, the use of a single platform for both T cell enrichment andexpansion simplifies and improves on existing protocols.

Tumor-specific T cells can now be generated by genetic engineering oflymphocytes to express anti-cancer TCR or chimeric antigen receptors(CAR)¹ and are a promising approach to increasing availability; however,the use of foreign receptors that have not been modulated by endogenoustolerance mechanisms theoretically increases the likelihood ofcross-reactivity and toxicity, with significant toxicities observed intrials³⁴. Autologous melanoma-infiltrating lymphocytes have provenhighly effective in clinical trials, but cannot be cultured from allmelanoma patients or for most other cancers².

A reliable method for generating responses from endogenous, naive cellscould thus increase both availability and safety³. Existing protocolshave demonstrated encouraging results with responses derived from naivecells^(35,36), but rely on repeated stimulation and cloning over manyweeks to months to generate between 10⁸ and 10¹⁰ tumor-reactive Tcells³⁶⁻³⁸ administered per infusion, leading to high cost andcomplexity. E+E is a promising novel approach, both in terms of totalnumber and purity, compared to existing attempts to generate robustexpansion in a shorter period. For example, expansion of NY-ESO1 withdendritic cell-based approaches after one week in culture is eitherundetectable or not reported, making the achievement of antigen specificpurities between 4-27% with 1000-fold expansion all the more remarkable.In contrast, several groups have reported effective expansion of thehigh precursor frequency MART1 response. For example, the novel DC-basedACE-CD8 platform described by Wölfl et al³⁹ is a well-characterizedsystem that has been very useful in helping define and optimizerequirements for expansion of human CD8 cells and shows an impressive 10day expansion, suggesting that further optimization of cultureconditions is required to support optimal MART1 expansion in vitro.Nevertheless, to generate the amount of T cells which may be neededclinically, the DC-based ACE-CD8 platform would still requirepotentially multiple plasmapherises from patients to generate weeklycultures of DC for expansion or use of non-antigen-specific techniques.Nano-aAPC based E+E is not subject to such constraints.

Assuming tumor-specific T cell precursor frequencies of approximately1-10 per million, approximately 0.5×10¹⁰ CD8 T cells harvested from asingle leukapharesis, and 1000-5000 fold expansion observed with E+E,more than 10⁸ antigen specific T cells could be generated in one week.Simultaneously expanding multiple antigens would increase this numberfurther, yielding sufficient cells for infusion. Thus, by eliminatingthe need to culture cellular APCs and streamlining the generation oflarge numbers of high-frequency tumor-specific T cells,Enrichment+Expansion can be a powerful addition to autologous tumorimmunotherapy protocols.

Methods

Mice and reagents. Pmel TCR/Thy1a Rag−/− transgenic mice were maintainedas homozygotes. C57BL/6j and Balb/C mice were purchased from JacksonLaboratories (Bar Harbor, Me.). All mice were maintained according toJohns Hopkins University's Institutional Review Board. Fluorescentlylabeled monoclonal antibodies were purchased from BioLegend (San Diego,Calif.).

Preparation of MHC-Ig Dimers and Nano-aAPC. Soluble MHC-Ig dimers,Kb-Ig, Db-Ig, and A2-Ig were prepared and loaded with peptides asdescribed.¹⁹ Nano-aAPC were manufactured by direct conjugation of MHC-Igdimer and anti-CD28 antibody (37.51; BioLegend) to MACS Microbeads(Miltenyi Biotec) as described.¹⁵

Lymphocyte Isolation. Mouse lymphocytes were obtained from homogenizedmouse spleens and lymph nodes followed by hypotonic lysis of RBC.Cytotoxic lymphocytes were isolated using a CD8 no-touch isolation kitand magnetic enrichment column from Miltenyi Biotec (Cologne, Germany)following the manufacture's protocol. Where applicable, cells werelabeled with carboxyfluorescein succinimidyl ester (CFSE) for 15 minutesat 37° C., then washed extensively. For human studies, the ethicalcommittee of the Johns Hopkins University approved this study and allhealthy volunteers gave written informed consent. PBMC of HLA-A2+ donorswere obtained by density gradient centrifugation (Lymphocyte SeparationMedium PaqueFicoll-Paque®, GE Healthcare). Subsequently, CD8+ T cellswere isolated using a CD8 no-touch isolation kit and magnetic enrichmentcolumn from Miltenyi Biotec (Cologne, Germany) following themanufacture's protocol. Purified CD8+ T cells were stained withnano-aAPC for 1 h at 4° C. and enriched magnetically utilizingMS-columns (Miltenyi Biotec).

Enrichment and Expansion. 10 million CD8-enriched lymphocytes wereincubated with 10 μl of nano-aAPC for 1 hour at 4° C. Cell-particlemixtures were subsequently passed through a magnetic enrichment column,the negative fraction was collected and the positive fraction waseluted. Isolated fractions were mixed and cultured in 96 well roundbottom plates for 7 days in complete RPMI-1640 medium supplemented with10% human autologous serum and 3% T cell growth factor (TCGF) in 96-wellround-bottom plates (Falcon) in a humidified incubator providing 5% CO₂and 37° C. for 1 week. Medium and TCGF were replenished once a week.Specificity of CTL was monitored on day 0 and 7, by tetramer and MHC-Igstain utilizing FACS analysis.

Bystander In Vivo Experiments. Mixtures of pmel and wild-type B6 CD8+ Tlymphocytes were mixed at the indicated ratios. Cell mixtures werecultured for one week with 20 μl of Db-gp100 nano-aAPC prior to adoptivetransfer. Transient lymphopenia was induced in host mice by sublethalirradiation (500 cGy) one day before adoptive transfer with a MSDNordion Gammacell dual Cs137 source (Johns Hopkins Molecular ImagingCenter) in the indicated groups. Mice were treated both the day of andthe day after adoptive transfer with 30,000 units intraperitoneal IL-2.Seven and twenty-days after adoptive transfer, three mice per group weresacrificed and lymphocytes were isolated from peripheral blood, spleen,and inguinal, cervical, and axillary lymph nodes, and then stained withanti-Thy1.1 antibody.

Tumor Rejection Experiments. Tumor rejection experiments were performedas above, except 3×10⁵ B16 melanoma cells were injected subcutaneouslyten days prior to adoptive T cell transfer. Transient lymphopenia wasinduced one day before adoptive transfer as described above. 10 millionnaive lymphocytes from each donor were used to generate antigen-specificcells for each tumor host (up to 3 hosts per donor), representingapproximately 2×10⁵ tumor-specific T cells generated and transferredafter one week of culture. Mice were treated both the day of and the dayafter adoptive transfer with 30,000 units intraperitoneal IL-2. Tumorgrowth was monitored at 2 day intervals using digital calipers. Micewere sacrificed once tumors reached 150 mm².

TABLE 1 Antigen-Specific T Cell Expansion. Estimated T cell precursorfrequencies per 10 million lymphocytes. Antigen-specific cells generatedfrom 10 million lymphocytes (with reference). Precursor Frequency (per10 Ag-Specific Fold Antigen million cells) Cells Expansion Kb-Trp210-100⁷ 130,000 ± 80,000  1,300-13,000x Db-gp100 10-100⁵ 35,000 ± 10,000  350-3,500x Kb-SIINF 20-350⁷ 150,000 ± 75,000    450-7,500x A2-NY-ESO1 36²⁷ 44,000 ± 21,000 1200x A2-MART1 1000²⁷ 83,000 ± 37,000  83x

Example 2 Expansion of Neo-Antigens

The tumor antigens described thus far are previously known “sharedantigens” derived from proteins that are over-expressed in tumors, andpresent on or shared between tumors from multiple patients. With theadvent of genome-wide sequencing, it has been shown that most cancerscontain clonal, non-synonymous single base pair substitutions that maybind to the patient's MHC, thereby opening up new avenues forimmunotherapy.²⁹ Subsequent analyses have reinforced this idea.³⁸⁻⁴⁵These “neo-antigens” have theoretical advantages over shared antigens astumor targets, such as greater specificity for tumor tissue andpotentially higher-affinity TCR-MHC interactions. However, the patternof mutation is unique in each cancer, and methods must be developed forrapid personalized identification and targeting of these neo-antigens.

To generate T cell responses against neo-antigens usingEnrichment+Expansion, we utilized published “mutomes” described for themouse melanoma line B16 and colon carcinoma line CT26.^(46,47) Briefly,genomic and transciptomic data sets were combined to identify expressedsingle base pair substitutions (FIG. 6A). Eight or nine flanking aminoacids upstream and downstream of each SBS were extracted in silico.These ˜17-amino acid sequences were then processed by NetMHC, analgorithm that predicts binding of peptides to human HLA as well asmouse MHC alleles using an artificial neural network⁴⁸. This algorithmpredicted amino acid neo-epitopes 8 to 10 amino acids in length for CT26and B16 (Table 2). Seven candidate peptides representing a wide range ofpredicted affinities, 2 from CT26 and 5 from B16, were synthesized andused to generate neo-epitope specific nano-aAPC. E+E with nano-aAPCbearing these neo-epitopes was then performed and evaluated with MHCmultimers at Day 7.

Antigen-specific populations from Day 7 cultures were identified forboth of the two CT26-derived candidate peptides tested (FPS and SAF).FIG. 6B shows representative Day 7 cognate MHC staining of Ld-FPS andLd-SAF activated samples. Peptides derived from the B16 mutome showedresponsive (Db-YTG) and non-responsive (Kb-LAY) staining patterns (FIG.6B); overall 2/5 peptides explored (Db-YTG and Kb-VDW) showed strongresponses, 2/5 showed moderate responses (Db-IAM and Db-RTF), and 1/5was non-responsive (Kb-LAY). Peptide affinity for MHC as predicted byNetMHC (Table 2) did not accurately predict E+E response; strongresponders YTG and VDW had low predicted affinities at 991 and 9066 nMrespectively, whereas the non-responder LAY and equivocal responder IAMhad high predicted affinities at 69 and 5 nM respectively. Overall, thetotal number of cells generated at Day 7 approximated those observedwith the shared antigens Db-GP100 and Ld-A5, ranging from 15,000-40,000(FIG. 6C), but was less than the shared antigens Kb-TRP2 and Kb-SIIN.

TABLE 2 Candidate Neo-Epitopes Best Proteins for Mutant PeptidePredicted Peptides (includes Affinity (bold = 10 mer) 10 mers) (nM)Allele Actn4 VTFQAFIDV 210 H-2-Kb Atp11a QSLGFTYL 19 H-2-Kb Cpsf31RTFANNPGPM 2043 H-2-Db Dag1 TTTTKKARV 2024 H-2-Kb Ddb1 VLMINGEEV 153H-2-Db Ddx23 QTAMFTATM 112 H-2-Kb Dpf2 LALPNNYCDV 318 H-2-Db Eef2ESFAFTADL 277 H-2-Kb Fat1 IAMQNTTQL 5 H-2-Db Fzd7 VAHVAAFL 87 H-2-KbKif18b VDWENVSPEL 9066 H-2-Kb Mthfd11 TILNCFHDV 1761 H-2-Kb Orc2VVPSFSAEI 39 H-2-Kb Pbk AAVILRDAL 121 H-2-Db Plod2 VWQIFENPV 111 H-2-KbS100a132510039O18 TVVCTFFTF 379 H-2-Kb Sema3b VSAAQAERL 1487 H-2-KbTm9sf3 AIYHHASRAI 191 H-2-Kb Tnpo3 LAYLMKGL 69 H-2-Kb Tubb3 YTGEAMDEM991 H-2-Db Wdr82 TNGSFIRLL 87 H-2-KbList of candidate peptide sequences containing neo-epitopes derived fromB16 tumors, including MHC affinity as predicted by NetMHC.

REFERENCES FOR EXAMPLE 2 ONLY

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(7) Jenkins, M. K.; Moon, J. J. J. Immunol. 2012, 188, 4135-4140.

(8) Klebanoff, C. a; Gattinoni, L.; Palmer, D. C.; Muranski, P.; Ji, Y.;Hinrichs, C. S.; Borman, Z. a; Kerkar, S. P.; Scott, C. D.; Finkelstein,S. E.; Rosenberg, S. a; Restifo, N. P. Clin. Cancer Res. 2011, 17,5343-5352.

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(15) Perica, K.; De León Medero, A.; Durai, M.; Chiu, Y. L.; Bieler, J.G.; Sibener, L.; Niemöller, M.; Assenmacher, M.; Richter, A.; Edidin,M.; Oelke, M.; Schneck, J. Nanomedicine 2013, 10, 119-129. (16) Perica,K.; Tu, A.; Richter, A.; Bieler, J. G.; Edidin, M.; Schneck, J. P. ACSNano 2014.

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(26) Gattinoni, L.; Finkelstein, S. E.; Klebanoff, C. a; Antony, P. a;Palmer, D. C.; Spiess, P. J.; Hwang, L. N.; Yu, Z.; Wrzesinski, C.;Heimann, D. M.; Surh, C. D.; Rosenberg, S. a; Restifo, N. P. J. Exp.Med. 2005, 202, 907-912.

(27) Alanio, C.; Lemaitre, F.; Law, H. K. W.; Hasan, M.; Albert, M. L.Blood 2010, 115, 3718-3725. (28) Lu, X.; Jiang, X.; Liu, R.; Zhao, H.;Liang, Z. Cancer Lett. 2008, 271, 129-139.

(29) Cobbold, M.; Khan, N.; Pourgheysari, B.; Tauro, S.; McDonald, D.;Osman, H.; Assenmacher, M.; Billingham, L.; Steward, C.; Crawley, C.;Olavarria, E.; Goldman, J.; Chakraverty, R.; Mahendra, P.; Craddock, C.;Moss, P. a H. J. Exp. Med. 2005, 202, 379-386.

(30) Yee, C.; Savage, P. a; Lee, P. P.; Davis, M. M.; Greenberg, P. D.J. Immunol. 1999, 162, 2227-2234.

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1. 1.-83. (canceled)
 84. A method for identifying a T cell antigen orcomponent of a T cell repertoire, comprising: providing a samplecomprising T cells from a subject, screening the T cells against alibrary of paramagnetic particles each comprising on their surface anMHC-peptide antigen-presenting complex and a lymphocyte costimulatoryligand, wherein the particles are from about 10 to about 500 nm indiameter, wherein each particle presents a candidate peptide antigen,magnetically activating T cells in the sample that are specific for thecandidate peptide antigens by incubating the particles with the samplein the presence of a magnetic field, separating cells associated withthe paramagnetic particles from cells not associated with theparamagnetic particles, culturing the cells associated with theparamagnetic particles, and identifying one or more candidate peptideantigens that cause activation of the subject's T cells, whereinactivation of the subject's T cells is identified by measuring cytokineexpression or intracellular signaling indicative of T cell activation.85. The method of claim 84, wherein the subject is a cancer patient. 86.The method of claim 85, wherein the candidate peptides comprise peptideantigens predicted from genetic analysis of the patient's cancer. 87.The method of claim 84, wherein activation of the subject's T cells isidentified by cytokine expression, measured by immunochemistry orRT-PCR.
 88. The method of claim 84, wherein selected peptide antigensare loaded onto artificial antigen presenting cells and administered tothe subject.
 89. The method of claim 84, wherein T cells that recognizethe identified antigens are enriched and expanded ex vivo, andadministered to the subject.
 90. The method of claim 84, wherein thesubject is suspected of having an immune disease, and the paramagneticparticles present a library of candidate peptides indicative ofautoimmune and/or immune disease.
 91. The method of claim 84, whereinthe subject is suspected of having an infectious disease, and theparamagnetic particles, present a library of candidate peptidesindicative of infectious diseases.
 92. The method of claim 84, whereinthe incubation of the particles with the sample on the magnetic columnis from 15 minutes to 5 hours.
 93. The method of claim 84, wherein the Tcells associated with the paramagnetic particles are cultured with theparamagnetic particles, and cytokine expression is quantified withinabout 15 to 48 hours in culture.
 94. The method of claim 84, wherein theT cells associated with the paramagnetic particles are cultured with theparamagnetic particles, and intracellular signaling quantified after 15minutes to 5 hours in culture.